The major barrier to treating or preventing Alzheimer's disease (AD) is its unknown etiology/pathogenesis. However, increasing evidence supports a role for mitochondrial dysfunction in the pathogenesis of AD. Most information on mitochondrial dysfunction has been obtained one protein at a time. It is the hypothesis of this proposal that changes in the mitochonrial proteome are an early event in the pathogenesis of neuron degeneration in AD and that these changes may be evaluated using quantitative proteomics. In preliminary studies, we applied 2-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC/MS/MS) and the isotope coded affinity tag (ICAT) method to identify and quantify protein levels in mitochondria isolated from early AD, late stage AD and age-matched control subjects. 2D-HPLC/MS/MS analysis of ICAT-labeled mitochondrial samples identified and quantified 22 unique proteins. Our results show that for early AD subjects ATP synthase and pyruvate kinase (essential for ATP production) and VDAC (part of the permeability transition pore) are significantly depleted. In late stage AD subjects, our results show that most proteins involved in ATP production are significantly depleted along with VDAC while uracil DNA glycosylase (base excision repair) and guanine nucleotide-binding protein (extra-matrix signal modulation) are significantly increased. To test the hypothesis that changes in mitochondrial proteome are an early event in the pathogenesis of neuron degeneration in AD we will a) quantitatively evaluate mitochondrial protein expression in the hippocampus, temporal pole, superior and middle temporal gyrus and cerebellum of control, early AD and late-stage AD subjects, b) immunohistochemically examine tissue sections from the same subjects to determine if proteins with altered expression correlate with neuropathologic features, and c) quantitatively evaluate changes in mitochondrial protein expression in primary rat cortical neuron cultures resulting from AD relevant insults including AB, iron/hydrogen peroxide, glucose deprivation, and glutamate. [unreadable] [unreadable]